in a Fluorescence spectrometer i have to measure the emission that peak will saturated why? how to solve this problem? here with am send it one modal graph.
Saturation of the detector can be reduced by reducing the intensity of fluorescent light reaching the detector which in turn can be done by
- reducing the concentration of sample (solution samples I assume) in the solution
- reducing slit widths
- reducing detector sensitivity etc.
It could also be second order scatter. What is your excitation wavelength?
Hi Vincent,
The saturated peak you are seeing is almost certainly not fluorescence emission from your sample, rather it is due to second-order scattering, i.e. scattering at 2x the excitation wavelength.
You can reduce this by reducing your slit widths but you still will likely not get rid of it. You can also reduce this by increasing the absorbance of your sample at the excitation wavelength, but then you will run into problems with primary and secondary inner filter effects (and possibly even more scattering if the solution becomes turbid), so it is best to keep the absorbance at the excitation wavelength below 0.2.
The best option is to only collect emission up to about 10-20 nm before 2x the excitation wavelength, in your case stop collecting emission at 600 nm.
Not even sure if there is a fluorescent sample at all. Sometimes it is helpful for strongly scattering samples to change geometrie of light path vs. sample position, or use front-face illumination for a scattering solution, or back illumination for a fluorescent film, or whatever. This depends much on your sample type and composition.
To avoid straylight of your excitation source you can,should use a low-cost cut-off filter
Forgot to mention that this filter, a glass plate for instance to cut off the UV, must be in the detection line
I agree entirely with Marvin.
This is scattering light and can be reduced but not eliminated. Yes, the best way is to stop collection before the scattering peak, 550-600 nm should be fine.
Scattering light shifts with changing the excitation wavelength while fluorescence does not. So it can be easily determined that is is indeed scattering light.
Also you could just plot the graph you have between 330-550 nm, and set Y-scale between 0-100 so that the actual features become visible.
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